Schematic flow diagram representing a method of nested-PCR followed by Invader assay.

<p>(A) The initial PCR was performed to amplify a fragment of 386 bp length containing a part of the NS5A region from cDNA reverse-transcribed from HCV-RNA which was extracted from the serum of a patient. (B) An aliquot of the inital PCR product was used for the second PCR to amplify a 308 bp fragment. The second PCR product was diluted with water and subjected to the Invader assay. (C) Invader oligonucleotide and allele-specific probes anneal with target to form one base overlap. When the base is complementary to the opposing base in the allele-specific probe, cleavase recognizes the structure and releases 5’ flap. The released flap anneals to a FRET probe. The second cleavage reaction releases fluorophore resulting in the generation of a fluorescent signal. The Invader assay was done in triplicate. (D) Three types of standard nucleotide (Std-YY, Std-YH, and Std-HH) were prepared, which includes binary target sequences, corresponding to wild (Y) or mutant (H) variants, and annealing sites with the internal primers at each end. Tenfold serial dilusions of each standard were subjected to the second PCR. (E) The Invader assay for each standard was also performed in triplicate.